In recent years, most pharmaceutical companies have focused on the development of monoclonal antibodies (mAbs). Increasing upstream titers and shrinking development timelines have posed several challenges to downstream process development of mAbs. Some of the major strategies and tools to address these challenges include the development of highly efficient platforms, high-throughput screening (HTS) tools, and reduction of the number of unit operations to help with facility fit. In the future, mAbs may represent a smaller percentage of the pipeline as portfolios concentrate more on development of antibody fragments, nanobodies, biosimilar protein therapeutics, conjugated proteins and vaccines, Fc-fusions, and nonantibody protein scaffolds. In addition to high cell density mammalian expression, expanded utilization of other expression systems such as microbial and yeast will support these newer biotherapeutics (BioTx). The diversity of BioTx and expression systems will pose unique challenges for downstream development. Novel tools, approaches, and/or platforms will be required to enable rapid development. Furthermore, with the increasing emphasis on Quality by Design (QbD), there is a need to develop paradigms to apply QbD not only to mAbs but also to other BioTx.

Early biotechnology products in the US and Europe were characterized by low titers and low cell densities using various cell hosts (1, 2). Most companies had few recombinant products licensed or in development, including insulin, somatotropin, interferon, tissue plasminogen activator, erythropoietin, Factor VIII, and Factor IX. There were no benefits or need for platform development or expanding manufacturing capabilities. Cell culture was performed using numerous methods, including roller bottles and stirred tank bioreactors operated in perfusion, batch re-feed, or fed-batch mode (1).

Starting in 1986 with the licensure of Orthoclone Okt3 (muromonab-CD3), monoclonal antibodies (mAbs) such as Rituxan (rituximab), Herceptin (Trastuzumab), and Remicade (infliximab) have dominated the BioTx market (3). Most company pipelines saw a dramatic increase in the number of therapeutic mAb candidates, which in turn resulted in a desire to shorten development timelines. Due to the large doses required, antibodies are typically expressed at high titers in high cell density mammalian cell culture processes. This past decade has seen both the titers and cell densities increase by an order of magnitude to meet the clinical and commercial demands (4–6). Further, companies have made significant investments in manufacturing facilities, resulting in cell culture bioreactor capacities of 12,000–20,000L. The increasing volumes, cell densities, and protein masses have posed numerous new challenges for downstream processing.

ADDRESSING CHALLENGES IN mAb PROCESSING

Shrinking timelines have necessitated developing tools for faster process development. Because of the similarity of mAbs, which mainly differ from each other in their complementarity determining regions (CDR), these biomolecules are amenable to platform development and manufacturing processes. The standardization of cellular expression systems, bioreactor conditions, purification processes, manufacturing hardware, and disposables has resulted in faster clinical development and lower costs. Most companies have developed standardized purification processes typically consisting of a cell harvest method followed by a capture chromatographic step and one or polishing steps. The approach used at Pfizer has been described elsewhere (7–12). Weak partitioning chromatography (WPC) and a high throughput screening (HTS) tools have been essential for development of a robust two-column platform purification process. The benefits of a two-column purification process include reduced capital, footprint, quality systems, validation, cleaning, development costs, solutions, and water consumption. The HTS method has also been applied to the development of other purification processes for unique biotherapeutic modalities where screening of a large number of resins may be required.

Figure 1: mAb total mass-throughput and volumetric flux through the Viresolve Pro filter as a function of time. An X0HC and a Viresolve Pro prefilter were used with 93 g/L mAb in 3 mM histidine, pH 5.0 at 35 °C.

Another consideration in mAb processing is reducing the cost of goods (COGs). The cost of the virus-retaining filter, which can approach that of the protein A resin, can be reduced by using prefilters or optimizing protein concentration, temperature, buffer composition, and solution pH (see Figure 1) (13). The costs of freezing and storing bulk drug substance can be reduced by targeting high concentrations. Concentrations above 250 g/L have been achieved using ultrafiltration at elevated temperatures (14) and over 500 g/L using a wet-ultrafiltration membrane evaporation method (15) (see Figure 2).

Figure 2: Viscosity versus concentration for an Fc-fusion protein and a mAb concentrated using a wet-ultrafiltration membrane evaporation method.

A significant consequence of the transfer of products between facilities is the generation of in-process pools that exceed the capacity of the storage vessels. Linking unit operations through tandem processes would eliminate this constraint and additionally may reduce process time, process costs, and documentation requirements. Feasibility has been demonstrated for a tandem downstream process for the purification of mAbs employing an affinity Protein A capture step followed by a flow-through anion-exchange (AEX) step and a virus filtration step (VRF) (16).